Protein chromophore interactions play a major role in a variety of biologically relevant processes including the absorption of light by antenna proteins in photosynthesis, the fluorescent and phosphorescent properties of a variety of aquatic species and the visual perception system. Protein/chromophore complexes have also found wide and essential utility in biological imaging as fluorescent protein fusion tags. A fundamental and applicable understanding of these interactions is therefore of broad interest to a variety of field. Using as inspiration the natural system that gives rise to color vision in higher primates, we will create novel protein/chromophore systems and use them to fully interrogate the unique interplay between protein and chromophore that gives rise to the unique spectroscopic characteristics of the complex. We will then further develop these systems for real world applications in protein visualization and in vivo pH sensing. We have chosen the color vision system for inspiration because here nature has demonstrated that the absorption properties of a single ligand chromophore, 11-cis-retinal, can be modulated by its protein environment to cover the entire visual spectrum. In previous studies we have demonstrated that the absorption spectrum of protein-bound retinal can be modulated over the entire visual spectrum by the application of rational protein design strategies. This has been accomplished by controlling the electrostatic environment that surrounds the chromophore when embedded in the protein environment. We will now take these insights and apply them to the rational design of protein/fluorophore complexes that will modulate the emission spectrum of fluorophores. We will focus on solvatochromic fluorophores, because their emission spectra are exquisitely sensitive to the solvent polarity. The factors that govern the pKa of the chromophore will also be rigorously investigated by producing protein/chromophore complexes with a wide range of pKa values. The principles developed in these studies will be applied to produce a new class of colorimetric and fluorescent fusion proteins. These proteins will exhibit broad ranges of color, fluorescence excitation and emission. A variety of fluorescent ligands will be designed, synthesized and tested to further optimize the system. These proteins will then be tested as fusion proteins both in vitro and in vivo in bacterial and mammalian cell types. Fluorescent pH sensors will be developed for in vivo measurement of pH in a variety of cellular milieus. These fusion proteins will be useful in a variety of applications including anaerobic environments. They will also be useful for temporal control of protein detection, where only the protein expressed in a defined time window will be detected. Together they will represent a complementary set of tools to the fluorescent proteins currently available and will extend the usefulness of this visualization technique substantially.